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From Proteins to Proteomes: Large Scale Protein Identification by Two-Dimensional Electrophoresis and Arnino Acid Analysis

Bio/Technology volume 14, pages 61–65 (1996)Cite this article

Abstract

Separation and identification of proteins by two-dimensional (2-D) electrophoresis can be used for protein-based gene expression analysis. In this report single protein spots, from polyvinylidene difluoride blots of micropreparative E. coli 2-D gels, were rapidly and economically identified by matching their amino acid composition, estimated pI and molecular weight against all E. coli entries in the SWISS-PROT database. Thirty proteins from an E. coli 2-D map were analyzed and identities assigned. Three of the proteins were unknown. By protein sequencing analysis, 20 of the 27 proteins were correctly identified. Importantly, correct identifications showed unambiguous “correct” score patterns. While incorrect protein identifications also showed distinctive score patterns, indicating that protein must be identified by other means. These techniques allow large-scale screening of the protein complement of simple organisms, or tissues in normal and disease states. The computer program described here is accessible via the World Wide Web at URL address (http://expasy.hcuge.ch/).

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References

  1. Wilkins, M. R., Sanchez, J.-C., Gooley, A. A., Appel, R. D., Humphery-Smith, I., Hochstrasser, D. F. and Williams, K. L. 1995. Progress with Proteome Projects: why all proteins expressed by a genome should be identified and how to do it. Biotech. Gen. Eng. Rev. In press.

  2. Wasinger, V. C., Cordwell, S. J., Poljak, A., Yan, J. X., Gooley, A. A., Wilkins, M. R., Duncan, M., Hairis, R., Williams, K. L. and Humphery-Smith, I. 1995. Progress with Gene-Product Mapping of the Mollicutes: Mycoplasma genitalium. Electrophoresis 16: In press.

    Article  CAS  Google Scholar 

  3. Garrels, J. I., Franza, B. R., Chang, C. and Latter, G. 1990. A Quantitative exploration of the REF52 protein database: cluster analysis reveals the major protein expression profiles in responses to growth regulation, serum stimulation, and viral transformation. Electrophoresis 11: 1114–1130.

    Article  CAS  Google Scholar 

  4. Celis, J. E. and Olsen, E. 1994. A qualitative and quantitative protein database approach identified individual and groups of functionally related proteins that are differentially regulated in simian virus 40 (SV40) transformed human keratinocytes: an overview of the functional changes associated with the transformed phenotype. Electrophoresis 15: 309–344.

    Article  CAS  Google Scholar 

  5. Garrels, J. I., Futcher, B., Koboyashi, R., Latter, G. I., Schwender, B., Volpe, T., Warner, J. R. and McLaughlin, C. S. 1994. Protein identification for a Saccharomyces cerevisiae protein database. Electrophoresis 15: 1466–1486.

    Article  CAS  Google Scholar 

  6. VanBogelen, R. A., Sanker, P., Clark, R. L., Bogan, J. A. and Neidhardt, F. C. 1992. The gene-protein database of Escherichia coli: edition 5. Electrophoresis 13: 1014–1054.

    Article  CAS  Google Scholar 

  7. Corbett, J. M. and Wheeler, C. H. and Baker, C. S. and Yacoub, M. H. and Dunn, M. J. 1994. The human myocardial two-dimensional gel protein database: update 1994. Electrophoresis 15: 1459–1465.

    Article  CAS  Google Scholar 

  8. Celis, J. E., Rasmussen, H. H., Olsen, E., Madsen, P., Leffers, H., Honore, B. and Dejgaard, K., Gromov, P., Hoffmann, H. J., Nielsen, M., Vassilev, A., Vintermyr, O., Hao, J., Celis, A., Basse, B., Lauridsen, H., Ratz, G. P., Andersen, A. H., Walbum, E., Kjaergaard, I., Puype, M., Van Damme, J. and Vandekerkhove, J. 1993. The human keratinocyte two-dimensional database: update 1993. Electrophoresis 14: 1091–1198.

    Article  CAS  Google Scholar 

  9. Hughes, G. J., Frutiger, S., Paquet, N., Pasquali, C., Sanchez, J.-C., Tissot, J.-D., Bairoch, A., Appel, R. D. and Hochstrasser, D. F. 1993. Human liver protein map: the 1993 update. Electrophoresis 14: 1216–1222.

    Article  CAS  Google Scholar 

  10. Latham, K. E., Garrels, J. I., Chang, C. and Solter, D., 1992. Analysis of embryonic mouse development: construction of a high resolution two-dimensional gel protein database. Appl. Theor. Electrophor. 2: 163–170.

    CAS  PubMed  Google Scholar 

  11. Ji, H., Whitehead, R. H., Reid, G. E., Moritz, R. L., Ward, L. D. and Simpson, R. J. 1994. Twodimensional electrophoretic analysis of proteins expressed by normal and cancerous human crypts: application of mass spectrometry to peptide-mass fingerprinting. Electrophoresis 15: 391–405.

    Article  CAS  Google Scholar 

  12. Matsudaira, P. 1987. Sequence from picomole quantities of proteins electroblotted to polyvinylidene membranes. J. Biol. Chem. 21: 10035–10038.

    Google Scholar 

  13. Rosenfeld, J., Capdevielle, J., Guillemot, J. C. and Ferrara, P. 1992. In-gel digestion of proteins for internal sequence analysis after one- or two-dimensional gel electrophoresis. Anal. Biochem. 203: 173–179.

    Article  CAS  Google Scholar 

  14. Honore, B., Leffers, H., Madsen, P. and Celis, J. E. 1993. Interferon-gamma up-regulates a unique set of proteins in human keratinocytes. Molecular cloning and expression of the cDNA encoding the RGD-sequence containing protein IGUP I-5111. Eur. J. Biochem. 218: 421–430.

    Article  CAS  Google Scholar 

  15. Patterson, S. D. 1994. From electrophoretically separated proteins to identification: strategies for sequence and mass analysis. Anal. Biochem. 221: 1–15.

    Article  CAS  Google Scholar 

  16. Cottrell, J. S. 1994. Protein identification by peptide mass fingerprinting. Peptide Research 7: 115–124.

    CAS  PubMed  Google Scholar 

  17. Eckerskorn, C., Jungblut, P., Mewes, W, Klose, J. and Lottspeich, F. 1988. Identification of mouse brain proteins after two-dimensional electrophoresis and electroblotting by microsequence analysis and amino acid composition analysis. Electrophoresis 9: 830–838.

    Article  CAS  Google Scholar 

  18. Shaw, G. 1993. Rapid identification of proteins. Proc. Natl. Acad. Sci. USA 90: 5138–5142.

    Article  CAS  Google Scholar 

  19. Hobohm, U., Houthaeve, T. and Sander, C. 1993. Amino acid analysis and protein database compositional search as a rapid and inexpensive method to identify proteins. Anal. Biochem. 222: 202–209.

    Article  Google Scholar 

  20. Jungblut, P., Dzionara, M., Klose, J. and Wittman-Leibold, B. 1992. Identification of tissue proteins by amino acid analysis after purification by two dimensional electrophoresis. J. Prot. Chem. 11: 603–612.

    Article  CAS  Google Scholar 

  21. Golaz, O., Hughes, G. J., Frutiger, S., Paquet, N., Bairoch, A., Pasquali, C., Sanchez, J.-C., Tissot, J.-D., Appel, R. D., Walzer, C., Balant, L. and Hochstrasser, D. F. 1993. Plasma and red blood cell protein maps: the 1993 update. Electrophoresis 14: 1223–1231.

    Article  CAS  Google Scholar 

  22. Cordwell, S., Wilkins, M. R., Cerpa-Poljak, A., Gooley, A. A., Duncan, M., Williams, K. L. and Humphery-Smith, I. 1995. Cross-species identification of proteins separated by two-dimensional electrophoresis using MALDI-TOF and amino acid composition. Electrophoresis 16: 438–443.

    Article  CAS  Google Scholar 

  23. Bairoch, A. and Boeckmann, B. 1994. The SWISS-PROT protein sequence bank: current status. Nucl. Acids Res. 22: 3578–3580.

    Article  CAS  Google Scholar 

  24. Strupat, K., Karas, M., Hillenkamp, F., Eckerskorn, C. and Lottspeich, F. 1994. Matrix-assisted laser desorption ionization mass spectrometry of proteins electroblotted after polyacrylamide gel electrophoresis. Anal. Chem. 66: 464–470.

    Article  CAS  Google Scholar 

  25. Garrels, J. I. 1989. The QUEST system for quantitative analysis of two-dimensional gels. J. Biol. Chem. 264: 5269–5282.

    CAS  PubMed  Google Scholar 

  26. Weitzhandler, M., Kadlecek, D., Avdalovic, N., Forte, J. G., Chow, D. and Townsend, R. R. 1993. Monosaccharide and oligosaccharide analysis of proteins transferred to polyvinylidene fluoride membranes after sodium dodecyl sulfate-polyacrylamide gel electrophoresis. J. Biol. Chem. 268: 5121–5130.

    CAS  PubMed  Google Scholar 

  27. Packer, N., Wilkins, M. R., Lawson, M. Golaz, O., Redmond, J., Gooley, A. A., Hochstrasser, D. F. and Williams, K. L. 1995. Characterisation of human plasma glycoproteins separated by two-dimensional gel electrophoresis. Bio/Technology 1: 66–70.

    Google Scholar 

  28. Bjellqvist, B., Sanchez, J.-C., Pasquali, C., Ravier, F., Paquet, N., Frutiger, S., Hughes, G. J. and Hochstrasser, D. F. 1993a. Micropreparative 2-D electrophoresis allowing the separation of samples containing milligram amounts of proteins. Electrophoresis 14: 1375–1378.

    Article  CAS  Google Scholar 

  29. Hochstrasser, D. F., Patchornik, A. and Merril, C. R. 1988. Development of polyacrylamide gels that improve the separation of proteins and their detection by silver staining. Anal. Biochem. 173: 214–225.

    Google Scholar 

  30. Hochstrasser, D. F., Harrington, M., Hochstrasser, A.-C., Miller, M. J. and Merril, C. R. 1988. Methods for increasing the resolution of two-dimensional protein electrophoresis. Anal. Biochem. 173: 226–234.

    Google Scholar 

  31. Hochstrasser, D. F. and Merril, C. R. 1988. Catalyst systems for polyacrylamide gel polymerization and detection of proteins by silver staining. Appl. Theor. Electrophor. 1: 35–40.

    CAS  PubMed  Google Scholar 

  32. Jin, Y. and Cerletti, N. 1992. Western blotting of transforming growth factor beta-2: optimisation of the electrophoretic transfer. Appl. Theor. Electrophor. 3: 85–90.

    CAS  PubMed  Google Scholar 

  33. Sanchez, J.-C., Ravier, F., Pasquali, C., Frutiger, S., Paquet, N., Bjellqvist, B., Hochstrasser, D. F. and Hughes, G. J. 1992. Improving the detection of proteins after transfer to polyvinylidene difluoride membranes. Electrophoresis 13: 715–717.

    Article  CAS  Google Scholar 

  34. Bjellqvist, B., Hughes, G. J., Pasquali, C., Paquet, N., Ravier, R., Sanchez, J.-C., Frutiger, S. and Hochstrasser, D. F. 1993. The focusing positions of polypeptides in immobilized pH gradients can be predicted from their amino acid sequences. Electrophoresis 14: 1023–1031.

    Article  CAS  Google Scholar 

  35. Haynes, P. A., Sheumack, D., Kibby, J., and Redmond, J. W. 1991a. Amino acid analysis using derivatisation with 9-fluorenylmethyl chloroformate and reversed-phase high performance liquid chromatography. J. Chromatogr. 540: 177–185.

    Article  CAS  Google Scholar 

  36. Ou, K., Wilkins, M. R., Yan, J. X., Gooley, A. A., Fung, Y, Scheumack, D. and Williams, K. L. 1995. Improved high-performance liquid chromatography of amino acids derivatised with 9-fluorenylmethyl chloroformate. J. Chromatogr. A, In press.

  37. Appel, R. D., Bairoch, A. and Hochstrasser, D. F. 1994a. A new generation of information retrieval tools for biologists: the example of the ExPASy WWW server. Trends Biochem. Sci. 19: 258–260.

    Article  CAS  Google Scholar 

  38. Appel, R. D., Sanchez, J.-C., Bairoch, A., Golaz, O., Ravier, E, Pasquali, C., Hughes, G. J. and Hochstrasser, D. F. 1994b. The SWISS-2DPAGE database of two-dimensional polyacrylamide gel electrophoresis. Nucl. Acids Res. 22: 3581–3582.

    CAS  PubMed  Google Scholar 

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Author information

Authors and Affiliations

  1. Macquarie University Centre for Analytical Biotechnology, Macquarie University, Sydney, NSW, 2109, Australia

    Marc R. Wilkins, Keli Ou, Jun X. Yan, Andrew. A. Gooley & Keith L. Williams

  2. Central Clinical Chemistry Laboratory and Medical Computing Centre of the University of Geneva, CH 1211, Geneva, 14, Switzerland

    Christian Pasquali, Ron D. Appel, Olivier Golaz, Jean-Charles Sanchez, Graham Hughes & Denis F. Hochstrasser

  3. Department of Microbiology, University of Sydney, NSW, 2006, Australia

    Ian Humphery-Smith

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  1. Marc R. Wilkins
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Correspondence to Keith L. Williams.

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Wilkins, M., Pasquali, C., Appel, R. et al. From Proteins to Proteomes: Large Scale Protein Identification by Two-Dimensional Electrophoresis and Arnino Acid Analysis. Nat Biotechnol 14, 61–65 (1996). https://doi.org/10.1038/nbt0196-61

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